pKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli

• pKILLIN is a versatile positive-selection cloning vector based on KILLIN.
• pKILLIN employs the smallest toxic indicator gene with multiple cloning sites.
• pKILLIN can efficiently clone any DNA fragments, including PCR products.
• Express a gene of interest as a His-tagged fusion protein for easy purification
• pKILLIN may be used to create genomic or cDNA libraries.

The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.